Methods for treating Angelman syndrome and related disorders

ABSTRACT

Provided herein are methods of treating Angelman Syndrome and/or Prader-Willi syndrome, that include administering an effective amount of a T-type calcium channel antagonist to a subject in need of the treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage Application under 35 U.S.C. § 371 and claims the benefit of International Application No. PCT/US2016/058487, filed Oct. 24, 2016, which claims the benefit of U.S. Provisional Application No. 62/245,038, filed Oct. 22, 2015, the entire disclosures of which are incorporated herein by reference.

TECHNICAL FIELD

This disclosure relates to treatment of disease by administering pharmaceutical compounds. In particular, the disclosure relates to the treatment of Angelman Syndrome and Prader-Willi syndrome by administering a T-type calcium channel antagonist.

BACKGROUND

Angelman Syndrome is a genetic disorder caused by the deletion or inactivation of genes on the maternally inherited chromosome 15 with silencing through imprinting of the paternal copy of the gene. The syndrome is characterized by intellectual and developmental disability, sleep disturbance, seizures, jerky movements, and a happy demeanor with frequent laughter or smiling. Angelman Syndrome affects males and females in equal numbers, with a prevalence of about 1 in 15,000 of the general population.

Angelman Syndrome is caused by the loss of the maternal contribution to a region of chromosome 15, most commonly by deletion although uniparental disomy, translocation, or single gene mutation in that region. In the region of the chromosome that is understood to be critical for Angelman Syndrome, the maternal and paternal contribution express certain genes very differently due to sex-specific epigenetic imprinting through DNA methylation. In a normal individual, the maternal allele of the gene UBE3A, part of the ubiquitin pathway, is expressed and the paternal allele is specifically silenced in the developing brain. Angelman Syndrome results when the maternal contribution is lost or mutated. A related syndrome, Prader-Willi Syndrome, results if the paternal contribution is lost or mutated.

The most common genetic defect leading to Angelman Syndrome is maternal deletion of about 4 megabases in chromosomal region 15q11-13 causing an absence of UBE3A expression in the paternally imprinted brain regions. UBE3A codes for an E6-AP ubiquitin ligase. Mice that do not express maternal UBE3A show severe impairments in hippocampal memory formation including a deficit in a learning paradigm that involves hippocampus-dependent contextual fear conditioning. Maintenance of long-term synaptic plasticity in hippocampal area CA1 in vitro is also disrupted.

Symptoms of Angelman Syndrome include functionally severe developmental delay; speech impairment, with minimal or no use of words; movement and balance disorders, including ataxia of gait and/or tremulous movement of limbs, frequent laughter/smiling; apparent happy demeanor; easily excitable personality, often with hand flapping movements; hypermotoric behavior; and short attention span. Frequently observed symptoms (in 80% or more of cases) include delayed, disproportionate growth in head circumference, seizures and abnormal electroencephalogram (EEG). In the EEG, three distinct interictal patterns are seen in Angelman Syndrome, including a very large amplitude 2-3 Hz rhythm most prominent in prefrontal leads, a symmetrical 4-6 Hz high voltage rhythm, and a 3-6 Hz activity punctuated by spikes and sharp waves in occipital leads, which is associated with eye closure. In addition, symptoms associated with Angelman Syndrome, occurring in about 20 to 80% of cases, include flat occiput, occipital groove, protruding tongue, feeding problems, prognathia, wide mouth, drooling, mouthing behaviors, hypopigmentation, hyper active lower extremity deep tendon reflexes, increased sensitivity to head, abnormal sleep patterns, fascination with water, abnormal food related behaviors, obesity, scoliosis, and constipation. Epilepsy typically occurs in 85% patients with Angelman Syndrome by age three, although development of seizures by age one occurs in less than 25% of patients. The types of seizures include atypical absences, generalized tonic-clonic seizures, atonic seizures and myoclonic seizures. Some patients experience multiple seizure types.

There is presently no cure available for Angelman Syndrome. Since Angelman Syndrome can result in multiple varieties of seizures, selection of appropriate anticonvulsant medications to treat epilepsy can be difficult. Angelman Syndrome affects sleep patterns, so melatonin may be used to promote sleep. Mild laxatives are also used frequently to encourage regular bowel movements. Beyond medication, physiotherapy is used to improve joint mobility and prevent stiffening of the joints. Speech and language therapy are commonly employed to address communication issues.

T-type calcium channels are low-voltage activated calcium channels that open during membrane depolarization and mediate calcium influx into cells after an action potential or depolarizing signal. T-type calcium channels known to be present within cardiac and smooth muscle, and also are present in many neuronal cells within the central nervous system. T-type calcium channels (transient opening calcium channels) are distinct from L-type calcium channels (Long-Lasting calcium channels) due to their ability to be activated by more negative membrane potentials, their small single channel conductance, and their non-responsiveness to traditional calcium channel antagonist drugs, targeting L-type calcium channels.

T-type calcium channels open following small membrane depolarizations. T-type calcium channels have been primarily studied in the context of neuronal and cardiomyocyte function, and have been implicated in hyperexcitability disorders, such as epilepsy and cardiac dysfunction. Voltage gated calcium channels are not generally expressed in non-excitable cells, but there is evidence that T-type calcium channels are expressed in cancer cells of non-excitable lineages.

T-type calcium channels are activated and inactivated by small membrane depolarizations, and display slow deactivation rates. Thus, these channels can carry depolarizing current at low membrane potentials and mediate cellular “window” currents, which occur within the voltage overlap between activation and steady state inactivation at low or resting membrane potentials. T-type calcium channels can maintain window current at non-stimulated or resting membrane potentials, thereby allowing a sustained inward calcium current carried by a portion of channels that are not inactivated. Mediation of window current allows T-type calcium channels to regulate intracellular calcium levels, both in electrically firing cells such as neurons, and in non-excitable tissues, under non-stimulated or resting cellular conditions.

Voltage-gated calcium channels are made up of several subunits. The α₁ subunit is the primary subunit that forms the transmembrane pore of the channel. The α₁ subunit also determines the type of calcium channel. The β, α₂δ, and γ subunits, present in only some types of calcium channels, are auxiliary subunits that play secondary roles in the channel. The al subunit is composed of four domains (I-IV), with each domain containing 6 transmembrane segments (S1-S6), and hydrophobic loops between the S5 and S6 segments of each domain form the pore of the channel. Sub-types of the T-type calcium channel are defined by the specific α₁ subunit as shown in Table 1.

TABLE 1 T-type Calcium Channel Sub-Types Designation α₁ subunit Gene Cav3.1 α₁G CACNA1G Cav3.2 α₁H CACNA1H Cav3.3 α₁I CACNA1I

Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II or CaMKII) is a serine/threonine-specific protein kinase that is regulated by the Ca′/calmodulin complex. CaMKII has 28 isoforms and is involved in many signaling cascades and may be a mediator of learning and memory and may play a role in homeostasis and reuptake in cardiomyocytes, chloride transport in epithelia, positive T-cell selection, and CD8 T-cell activation. Misregulation of CaMKII may be linked to Alzheimer's disease, Angelman syndrome, and heart arrhythmia. CaMKII is also implicated in long-term potentiation (LTP)—a molecular process of strengthening active synapses that may contribute to the processes of memory—and may therefore be important to memory formation.

The sensitivity of the CaMKII enzyme to calcium and calmodulin is governed by variable and self-associative domains. Initially, the enzyme is activated by binding to the calcium/calmodulin complex, which leads to phosphorylation of the Threonine 286 site and activation of the catalytic domain. Once activated, as greater amounts of calcium and calmodulin accumulate, autophosphorylation can occur leading to persistent activation of the CaMKII enzyme. Autophosphorylation is enhanced by the stacked ring structure of the holoenzyme: the close proximity of these rings allows for phosphorylation of neighboring CaMKII enzymes. Dephosphorylation of the Threonine 286 residue leads to inactivation of CaMKII.

SUMMARY

The present disclosure provides a method of treating Angelman Syndrome or Prader-Willi syndrome. The method includes administering to a subject in need of such treatment a therapeutically effective amount of a T-type calcium channel antagonist. Also provided is the use of -type calcium channel antagonist for treating Angelman Syndrome or Prader-Willi syndrome. The disclosure also provides the use of -type calcium channel antagonist in the manufacture of a medicament for treating Angelman Syndrome or Prader-Willi syndrome.

In some embodiments, the T-type calcium channel antagonist is a calcium channel antagonist that selectively targets T-type calcium channels.

In some embodiments, the T-type calcium channel antagonist is a small molecule.

In some embodiments, the T-type calcium channel antagonist is an antibody.

In some embodiments, the T-type calcium channel antagonist is a siRNA.

In some embodiments, the T-type calcium channel antagonist selectively targets CaV3.1.

In some embodiments, the T-type calcium channel antagonist selectively targets CaV3.2.

In some embodiments, the T-type calcium channel antagonist selectively targets CaV3.3.

In some embodiments, the T-type calcium channel antagonists antagonize a T-type calcium channel in a cell when the membrane potential of the cell is in the range from about −60 mV to about −30 mV, e.g., about −40 mV.

In some embodiments, T-type calcium channel antagonist is selected from the group consisting of mibefradil, diltiazem, nifedipine, nitrendipine, nimodipine, niludipine, niguldipine, nicardipine, nisoldipine, amlodipine, felodipine, isradipine, ryosidine, gallopamil, verapamil, tiapamil, pimozide, thioridazine, NNC 55-0396, TTL-1177, anandamide, pimozide, penfluridol, clopimozide, fluspirilene, haloperidol, droperidol, benperidol, triperidol, melperone, lenperone, azaperone, domperidone, antrafenine, aripiprazole, ciprofloxacin, dapiprazole, dropropizine, etoperidone, itraconazole, ketoconazole, levodropropizine, mepiprazole, naftopidil, nefazodone, niaprazine, oxypertine, posaconazole, trazodone, urpidil, vesnarinone, manidipine, nilvadipine, benidipine, efonidipine, flunarizine, anandamide, lomerizine, phenytoin, zonisamide, U-92032, tetralol, mibefradil, NNC 55-0396, TTA-A2, TTA-A8, TTA-P1, 4-aminomethyl-4-fluoropiperidine (TTA-P2), TTA-Q3, TTA-Q6, MK-5395, MK-6526, MK-8998, Z941, Z944, ethosuximide, phensuximide, mesuximide, desmethylmethsuximide, efonidipine, trimethadione, dimethadione, ABT-639, TTL-1177, KYSO5044, nickel, and kurtoxin, and combinations thereof.

In some embodiments, the T-type calcium channel antagonist is selected from the group consisting of mibefradil, efonidipine, TTL-1177, and nickel, and combinations thereof.

In some embodiments, the T-type calcium channel antagonist is mibefradil.

In some embodiments, the T-type calcium channel antagonist is MK-8998.

In some embodiments, the disease is Angelman syndrome.

In some embodiments, the disease is Prader-Willi syndrome.

In some embodiments, the treatment comprises reducing or ameliorating a neurological symptom, which can be one or more of hyperactivity, abnormal sleep pattern, seizure, and ataxia.

In some embodiments, the treatment comprises reducing the frequency of seizure in the subject.

In some embodiments, the treatment comprises reducing the severity of seizure in the subject.

In some embodiments, the treatment comprises reducing the frequency of dystonia in the subject.

In some embodiments, the treatment comprises reducing the severity of dystonia in the subject.

In some embodiments, the treatment comprises reducing the frequency of ataxia in the subject.

In some embodiments, the treatment comprises reducing the severity of ataxia in the subject.

In some embodiments, the treatment comprises reducing the frequency of tremor, e.g., tremulous movement of the limbs, in the subject.

In some embodiments, the treatment comprises reducing the severity of tremor, e.g., tremulous movement of the limbs, in the subject.

In some embodiments, the treatment comprises reducing the severity of ataxia in the subject.

In some embodiments, the treatment comprises improving cognition or reducing cognitive deficits in the subject.

In some embodiments, the treatment comprises improving memory or reducing memory deficits in the subject.

In some embodiments, the treatment comprises improving attention or reducing attention deficits in the subject.

In some embodiments, the treatment comprises reducing obesity in the subject.

In some embodiments, the treatment comprises reducing the amount or rate of weight gain in the subject.

In some embodiments, the treatment comprises reducing body weight in the subject.

In some embodiments, the treatment comprises reducing hunger or increasing satiety in the subject.

In some embodiments, the selective T-type calcium channel modulator substantially crosses the blood brain barrier. In other embodiments, the T-type calcium channel modulator does not substantially cross the blood brain barrier.

In some embodiments, the treatment includes administering to the subject an additional therapeutic agent, which can be, e.g., an additional T-type calcium channel inhibitor or an anticonvulsive agent.

In some embodiments, the treatment includes administering an additional therapy, which can be selected, e.g., from the group consisting of physical therapy, occupational therapy, communication therapy, and behavioral therapy.

The disclosure provides a method of treating a disease or disorder associated with CaMKII autophosphorylation. The method includes administering to a subject in need of such treatment a therapeutically effective amount of a T-type calcium channel antagonist. Also provided is the use of -type calcium channel antagonist for treating a disease or disorder associated with CaMKII autophosphorylation. The disclosure also provides the use of -type calcium channel antagonist in the manufacture of a medicament for treating a disease or disorder associated with CaMKII autophosphorylation.

In some embodiments, the disease or disorder is Angelman Syndrome or Prader-Willi syndrome.

In some embodiments, the T-type calcium channel antagonist is a calcium channel antagonist that selectively targets T-type calcium channels.

In some embodiments, the T-type calcium channel antagonist is a small molecule.

In some embodiments, the T-type calcium channel antagonist is an antibody.

In some embodiments, the T-type calcium channel antagonist is a siRNA.

In some embodiments, the T-type calcium channel antagonist selectively targets Cav3.1.

In some embodiments, the T-type calcium channel antagonist selectively targets Cav3.2.

In some embodiments, the T-type calcium channel antagonist selectively targets Cav3.3.

In some embodiments, the T-type calcium channel antagonists antagonize a T-type calcium channel in a cell when the membrane potential of the cell is in the range from about −60 mV to about −30 mV, e.g., about −40 mV.

Also provided is method of treating obesity in a subject in need thereof, comprising administering to the subject a T-type calcium channel antagonist.

In some embodiments, the treatment comprises reducing body weight in the subject.

In some embodiments, the treatment comprises reducing the amount or rate of weight gain in the subject.

In some embodiments, the treatment comprises reducing body weight in the subject.

In some embodiments, the treatment comprises reducing hunger or increasing satiety in the subject.

In some embodiments, the T-type calcium channel antagonist is a calcium channel antagonist that selectively targets T-type calcium channels.

In some embodiments, the T-type calcium channel antagonist is a small molecule.

In some embodiments, the T-type calcium channel antagonist is an antibody.

In some embodiments, the T-type calcium channel antagonist is a siRNA.

In some embodiments, the T-type calcium channel antagonist selectively targets Cav3.1.

In some embodiments, the T-type calcium channel antagonist selectively targets Cav3.2.

In some embodiments, the T-type calcium channel antagonist selectively targets Cav3.3.

In some embodiments, the T-type calcium channel antagonists antagonize a T-type calcium channel in a cell when the membrane potential of the cell is in the range from about −60 mV to about −30 mV, e.g., about −40 mV.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Where the first page number of a reference is given in a citation, it is to be understood that reference is being made to the entire article cited. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows results of audiogenic-induced epilepsy tests using UBE3/129sv mice treated with mibefradil (10 mg/kg, 40 mg/kg) or MK-8998 (also known as “CX-8998”) (10 mg/kg, 30 mg/kg, 60 mg/kg).

DETAILED DESCRIPTION

The present disclosure describes that T-type voltage-gated calcium channels are involved in Angelman Syndrome. The present disclosure further describes that modulation of such T-type voltage-gated calcium channels can be effective for the treatment of Angelman Syndrome and related conditions such as Prader-Willi Syndrome.

While not being limited by any theory, a T-type calcium channel antagonist as described herein (e.g., mibefradil) can inhibit the inhibitory autophosphorylation of Ca²⁺/calmodulin-dependent protein kinase II (i.e., CaMKII), which is a serine-threonine-specific protein kinase that is regulated by the Ca²⁺/calmodulin complex and is involved in various signaling cascades. Neuronal depolarization increases levels of CaMKII autophosphorylation and chelation of extracellular calcium robustly decreases basal CaMKII autophosphorylation and increases levels of total CaMKII in cytosolic fractions. It has been shown that inhibition of T-type calcium channels with mibefradil (5 μM) or NiCl₂ (100 μM) significantly decreases threonine autophosphorylation of CaMKIIα by 37% or 35% respectively, demonstrating that basal CaMKIIα activation and autophosphorylation in the striatum is at least partially supported by calcium influx through T-type calcium channel inhibitors. Pasek et al., Mol. Cell. Neurosci., 2015, 68. 234-43.

Dysregulation of CaMKII is associated with neurological disorders including, but not limited to, Angelman Syndrome, Prader-Willi Syndrome, cerebral ischemia, and Alzheimer's disease, and it has been found that maintenance of basal levels of CaMKII autophosphorylation requires T-type calcium channel activity. It is considered that that blocking T-type calcium channels can block the calcium from binding to CamKII, reducing inhibitory autophosphorylation and thus provide increased activation of CamKII and beneficial therapeutic effects.

Accordingly, the present application provides T-type calcium channel antagonists that can inhibit autophosphorylation of CaMKII and/or inhibit T-type calcium channels, which are useful for the treatment of disorders associated with dysregulation of CaMKII such as Angelman Syndrome.

I. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure belongs.

For the terms “e.g.” and “such as,” and grammatical equivalents thereof, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise.

The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

The term “about” means “approximately” (e.g., plus or minus approximately 10% of the indicated value).

The term “small molecule” means an organic compound with a molecular weight of about 1000 or less.

The term “subject,” referring to the subject of treatment, means any animal, including mammals, e.g., human.

The phrase “therapeutically effective amount” refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response that is being sought in a tissue, system, animal, individual or human by a researcher, veterinarian, medical doctor or other clinician.

The term “treating” or “treatment” refers to one or more of (1) preventing a disease; e.g., preventing a disease, condition or disorder in an individual who may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease; (2) inhibiting a disease; e.g., inhibiting a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology); and (3) ameliorating a disease; for example, ameliorating a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology) such as decreasing the severity of disease or reducing or alleviating one or more symptoms of the disease.

The term “T-type calcium channel antagonists” refers to a substance that reduces the activity of T-type calcium channels, e.g., through binding to, or otherwise inhibiting or blocking activity of the channel, or through reducing the expression of T-type calcium channels.

The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination.

The following abbreviations and symbols may be used in the present disclosure: CaMKII (calcium/calmodulin-dependent protein kinase II); DNA (deoxyribonucleic acid); dsRNA (double stranded RNA); g (gram); IC₅₀ (half maximal inhibitory concentration); kg (kilogram); mg (milligram); mRNA (messenger RNA); RNA (ribonucleic acid); RNAi (RNA interference); siRNA (small interfering RNA), wt (weight).

II. Methods of Treatment

Provided herein are methods of treating a disease associated with dysregulation of CaMKII, dysregulation of T-type calcium channels, or a combination of dysregulation of CaMKII and dysregulation of T-type calcium channels, in a subject in need thereof. The subject can include mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, primates, and humans. In some embodiments, the subject is a human. In some embodiments, the treating comprises reducing or ameliorating a neurological symptom associated with dysregulation of CaMKII. In some embodiments, the neurological symptom is selected from the group consisting of mental retardation, cognitive dysfunction, deterioration of long-term potentiation (e.g., deterioration associated with Angelman Syndrome, Prader-Willi syndrome, or Alzheimer's disease), and ataxia. In some embodiments, the method comprises administering a therapeutically effective amount of a T-type calcium channel antagonist as described herein, or a pharmaceutically acceptable salt thereof, to a subject in need thereof to block inhibitory autophosphorylation of CaMKII.

The present disclosure further provides methods of treating a disease selected from Angelman Syndrome and Prader-Willi syndrome in a subject in need thereof. In some embodiments, the method comprises administering a therapeutically effective amount of a T-type calcium channel antagonist as described herein, to the subject in need of the treatment. In some embodiments, the disease is Angelman Syndrome. In some embodiments, the disease is Prader-Willi syndrome.

In some embodiments, the treatment comprises reducing or ameliorating a neurological symptom associated with Angelman Syndrome or Prader-Willi Syndrome. The neurological symptoms can include but are not limited, to any one or more of hyperactivity, abnormal sleep pattern, seizure, and ataxia. In some embodiments, the neurological symptom comprises seizure. As used herein, the term “seizure” includes, but is not limited to, and can be any one or more of absence seizures (e.g., typical and atypical absences), atonic seizures, catamenial seizures, cluster seizures, episodic seizures, Dravet syndrome (i.e., severe myoclonic epilepsy in infancy or SMEI), focal seizures (i.e., partial seizures), focal seizures with secondary generalization, focal seizures with secondary generalized clonic seizures, infantile spasmsmyoclonic seizures, tonic seizures, and tonic clonic seizures.

In some embodiments, the subject of treatment has symptoms that include hyperactivity. In some embodiments, the subject of treatment has symptoms that include abnormal sleep pattern. In some embodiments, the subject of treatment has symptoms that include seizure, including any one or more of the types of seizure defined above. In some embodiments, the subject of treatment has symptoms that do not include ataxia. In some embodiments, the subject of treatment has symptoms that do not include abnormal sleep pattern. In some embodiments, the subject of treatment has symptoms that do not include seizure, or that do not include one or more of atypical absence seizures, typical absences, atonic seizures, catamenial seizures, cluster seizures, episodic seizures, Dravet syndrome focal seizures (with or without secondary generalization or secondary generalized clonic seizures), infantile spasms, myoclonic seizures, tonic seizures, or tonic clonic seizures.

Also provided is method of treating obesity in a subject in need thereof, comprising administering to the subject a T-type calcium channel antagonist. In some embodiments, the treatment comprises reducing body weight in the subject. In some embodiments, the treatment comprises reducing the amount or rate of weight gain in the subject. In some embodiments, the treatment comprises reducing body weight in the subject. In some embodiments, the treatment comprises reducing hunger or increasing satiety in the subject.

The treatment can be administered at an effective dose for the particular compound. Examples of suitable doses include, in humans, include dosages in the range from about 1 mg to about 2000 mg, e.g., about 1 mg to about 2000 mg, about 2 mg to about 2000 mg, about 5 mg to about 2000 mg, about 10 mg to about 2000 mg, about 20 mg to about 2000 mg, about 50 mg to about 2000 mg, about 100 mg to about 2000 mg, about 150 mg to about 2000 mg, about 200 mg to about 2000 mg, about 250 mg to about 2000 mg, about 300 mg to about 2000 mg, about 400 mg to about 2000 mg, about 500 mg to about 2000 mg, about 1000 mg to about 2000 mg, about 1 mg to about 1000 mg, about 2 mg to about 1000 mg, about 5 mg to about 1000 mg, about 10 mg to about 1000 mg, about 20 mg to about 1000 mg, about 50 mg to about 1000 mg, about 100 mg to about 1000 mg, about 150 mg to about 1000 mg, about 200 mg to about 1000 mg, about 250 mg to about 1000 mg, about 300 mg to about 1000 mg, about 400 mg to about 1000 mg, about 500 mg to about 1000 mg, about 1 mg to about 500 mg, about 2 mg to about 500 mg, about 5 mg to about 500 mg, about 10 mg to about 500 mg, about 20 mg to about 500 mg, about 50 mg to about 500 mg, about 100 mg to about 500 mg, about 150 mg to about 500 mg, about 200 mg to about 500 mg, about 1 mg to about 250 mg, about 2 mg to about 250 mg, about 5 mg to about 250 mg, about 10 mg to about 250 mg, about 20 mg to about 250 mg, about 50 mg to about 250 mg, about 100 mg to about 250 mg, about 1 mg to about 100 mg, about 2 mg to about 100 mg, about 5 mg to about 100 mg, about 10 mg to about 100 mg, about 20 mg to about 100 mg, about 50 mg to about 100 mg. Doses can be, e.g., about 1 mg, about 2 mg, about 5 mg, about 10 mg, about 20 mg, about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 400 mg, about 500 mg, about 1000 mg, about 1500 mg, or about 2000 mg. Doses can be less than about 2000 mg, less than about 1500 mg, less than about 1000 mg, less than about 5000 mg, less than about 400 mg, less than about 250 mg, less than about 200 mg, less than about 150 mg, less than about 100 mg, less than about 50 mg, less than about 20 mg or less than about 10 mg. Each of the doses can be doses that are administered at a frequency of once daily, twice daily, three times daily or four times daily, or less than once daily. Each of the doses can also be the dose that is administered to an adult with equivalent (scaled) dose being administered for pediatric patients.

The dose can be a dose that provides a plasma level (e.g., a steady state or a maximum level) of about 100 ng/mL, about 200 ng/mL, 500 ng/mL, about 1 μg/mL, about 2 μg/mL, about 5 μg/mL, about 10 μg/mL, about 20 μg/mL, about 50 μg/mL, about 100 μg/mL, about 200 μg/mL, about 250 μg/mL, or about 500 μg/mL, about 1000 μg/mL, or in a range between these values, or a concentration that is less than these values.

In some embodiments, treatment is continued for a period of about 1 week or longer. In some embodiments, treatment is continued for a period of about 2 weeks or longer. In some embodiments, treatment is continued for a period of about 3 weeks or longer. In some embodiments, treatment is continued for a period of about 4 weeks or longer. In some embodiments, treatment is continued for a period of about 8 weeks or longer. In some embodiments, treatment is continued for a period of about 12 weeks, or about 13 weeks, or longer. In some embodiments, treatment is continued for a period of about 24 weeks, or 26 weeks, or longer. In some embodiments, treatment is continued for a period of about 6 months or longer. In some embodiments, treatment is continued for a period of about 12 months or longer. In some embodiments, treatment is continued for a period of about 18 months or longer. In some embodiments, treatment is continued for a period of about 24 months or longer.

III. T-Type Calcium Channel Antagonists

The T-type calcium channel antagonist used in any of the methods described herein, or any of the embodiments thereof, can be one or more of the T-type calcium channel agonists described below.

The T-type calcium channel antagonist can be an antagonist of human T-type calcium channels when the subject of treatment is a human.

The T-type calcium channel antagonist can be a small molecule. Example small molecule T-type calcium channel antagonists which may be used in the methods provided herein include, but are not limited to, mibefradil, diltiazem, nifedipine, nitrendipine, nimodipine, niludipine, niguldipine, nicardipine, nisoldipine, amlodipine, felodipine, isradipine, ryosidine, gallopamil, verapamil, tiapamil, pimozide, thioridazine, NNC 55-0396, TTL-1177, anandamide, benzazepine derivatives, diphenylbutylpiperidine derivatives (e.g., pimozide, penfluridol, clopimozide, and fluspirilene), butyrophenone derivatives (e.g., haloperidol, droperidol, benperidol, triperidol, melperone, lenperone, azaperone, and domperidone), and phenylpiperazine derivatives (e.g., antrafenine, aripiprazole, ciprofloxacin, dapiprazole, dropropizine, etoperidone, itraconazole, ketoconazole, levodropropizine, mepiprazole, naftopidil, nefazodone, niaprazine, oxypertine, posaconazole, trazodone, urpidil, and vesnarinone), dihydropyridine derivatives (e.g., manidipine, nilvadipine, benidipine, and efonidipine), flunarizine, anandamide, lomerizine, phenytoin, zonisamide, U-92032, tetralol, tetralol derivatives (e.g., mibefradil), mibefradil derivatives (e.g., NNC 55-0396 dihydrochloride), TTA-A2, TTA-A8, TTA-P1, 4-aminomethyl-4-fluoropiperidine (TTA-P2), TTA-Q3, TTA-Q6, MK-5395, MK-6526, MK-8998, Z941, Z944, succinimide anticonvulsant derivatives (e.g., ethosuximide, phensuximide, and mesuximide also known as methsuximide, N-desmethylmethsuximide also known as (alpha)-methyl-(alpha)-phenyl-succinimide), and efonidipine (e.g. (R)-efonidipine), trimethadione, dimethadione, ABT-639, TTL-1177, KYSO5044, kurtoxin. Any of the T-type calcium channel inhibitors can be in the form of a pharmaceutically acceptable salt. Structures of certain T-type calcium channel inhibitors are shown below:

In some embodiments, T-type calcium channel small-molecule modulators may be selected from the group consisting of those described in the patents and published patent applications listed in Giordanetto et al, “T-type calcium channels inhibitors: a patent review,” Expert Opin. Ther. Pat., 2011, 21, 85-101, including WO2004035000, WO9304047, WO2006098969, WO2009009015, WO2007002361, WO2007002884, WO2007120729, WO2009054982, WO2009054983, WO2009054984, US20090270413, WO2008110008, WO2009146539, WO2009146540, U.S. Pat. No. 8,133,998, WO2010083264, WO2006023881, WO2006023883, WO2005007124, WO2005009392, US2005245535, WO2007073497, WO200707852, WO2008033447, WO2008033456, WO2008033460, WO2008033464, WO2008033465, WO2008050200, WO20081 17148, WO2009056934, EP1568695, WO2008007835, KR754325, U.S. Pat. No. 7,319,098, US20100004286, EP1757590, KR2009044924, US2010094006, WO2009035307, US20090325979, KR75758317, WO2008018655, US20080293786, and US20100056545, each of which is incorporated by reference in its entirety.

In some embodiments, the T-type calcium channel antagonist is a small molecule. In some embodiments, the small molecule has a molecular weight of 1000 or lower, e.g., about 900 or lower, about 800 or lower, about 700 or lower, about 600 or lower, about 500 or lower, about 400 or lower, or in the range from about 100 to about 500, about 200 to about 500, about 200 to about 400, about 300 to about 400 or about 300 to about 500. In some embodiments, the T-type calcium channel antagonist is a selective T-type calcium channel antagonist. “Selective” in this context means that the T-type calcium channel antagonist is more potent at antagonizing T-type calcium channel calcium channels compared with other types of calcium channel, e.g., any one or more of L-type, N-type, P-type, Q-type and/or R-type calcium channels, e.g., compared with L-type calcium channels. Selectivity can be determined, e.g., by comparing the IC₅₀ of a compound in inhibiting T-type calcium channels with its IC₅₀ in inhibiting the other types of calcium channel: if the IC₅₀ for inhibiting T-type channels is lower than the IC₅₀ for inhibiting the other types of calcium channel, the compound is considered selective. An IC₅₀ ratio of 0.1 (or lower) denotes 10-fold (or greater) selectivity. An IC₅₀ ratio of 0.01 (or lower) denotes 100-fold (or greater) selectivity. An IC₅₀ ratio of 0.001 (or lower) denotes 1000-fold (or greater) selectivity. In some embodiments, the T-type calcium channel antagonist has selectivity for the T-type calcium channel that is 10-fold or greater, 100-fold or greater, or 1000-fold or greater compared with other types of calcium channel, e.g., any one or more of L-type, N-type, P-type, Q-type and/or R-type calcium channels, e.g., compared with L-type calcium channels.

In some embodiments, the T-type calcium channel antagonist is a selective T-type calcium channel inhibitor which is selected from the group consisting of phensuximide, methsuximide, methyl-phenyl-succinimide, R isomer of efonidipine, trimethadione, dimethadione, mibefradil, TTA-A2, TTA-A8, TTA-P1, TTA-P2, TTA-Q3, TTA-Q6, MK-5395, MK-6526, MK-8998, Z941, Z944, ABT-639, TTL-1177, KYSO5044, N C 55-0396 dihydrochloride, kurtoxin, or a derivative thereof.

In some embodiments, the T-type calcium channel antagonist is selected from the group consisting of mibefradil, efonidipine, TTL-1177, nickel, and combinations thereof.

In some embodiments, the T-type calcium channel antagonist is mibefradil.

In some embodiments, the T-type calcium channel antagonist is MK-5395.

In some embodiments, the T-type calcium channel antagonist is MK-6526.

In some embodiments, the T-type calcium channel antagonist is MK-8998.

In some embodiments, the T-type calcium channel antagonist is Z944.

In some embodiments, the T-type calcium channel antagonist can be other than ethosuximide.

In some embodiments, the T-type calcium channel antagonist can be a molecule that does not act as a pore-blocker of the T-type calcium channel. The T-type calcium channel antagonist can be, e.g., an allosteric inhibitor of T-type calcium channels.

In some embodiments, the T-type calcium channel antagonist can be one that does not substantially affect one or more sodium channels such as sodium channels having Nav 1.1, Nav 1.2, Nav 1.3, Nav 1.4, Nav 1.5, Nav 1.6, Nav 1.7, Nav 1.8, or Nav 1.9 alpha subunits, and/or Nav β1, Nav β2, Nav β3, Nav β4 subunits. The T-type calcium channel antagonist can be selective for T-type calcium channel compared to inhibition of sodium channels, e.g., having at least a 2-fold, at least a 5-fold, at least a 10-fold, at least a 20-fold, at least a 100-fold, at least a 500-fold or at least a 1000-fold selectivity (expressed, e.g., in terms of K_(i)). The T-type calcium channel inhibitor can be one that does not substantially decrease the non-inactivating sodium current in thalamocortical neurons, e.g., that decreases the inactivating sodium current by about 20% or less, about 10% or less, about 5% or less, about 2% or less, or about 1% or less.

In some embodiments, the T-type calcium channel antagonist can be one that does not substantially affect one or more potassium channels such as calcium activated potassium channels (BK channels, SK channels, IK channels), inwardly rectifying potassium channels (ROMK, GPCR regulated, ATP sensitive), tandem pore domain potassium channels (TWIK (TWIK-1, TWIK-2, KCNK7), TREK (TREK-1, TREK-2, TRAAK), TASK (TASK-1, TASK-3, TASK-5), TALK (TASK-2, TALK-1, TALK-2), THIK (THIK-1, THIK-2), TRESK), or voltage gated potassium channels (hERG, KvLQT). The T-type calcium channel antagonist can be selective for T-type calcium channel compared to inhibition of potassium channels, e.g., having at least a 2-fold, at least a 5-fold, at least a 10-fold, at least a 20-fold, at least a 100-fold, at least a 500-fold or at least a 1000-fold selectivity (expressed, e.g., in terms of K_(i)).

In some embodiments, the T-type calcium channel antagonist can be one that does not substantially affect one or more GABA receptors such as GABA_(A) receptors, GABA_(A-ρ) subclass (GABA_(C)) receptors, or GABA_(B) receptors. In some embodiments, the T-type calcium channel antagonist can be one that does not substantially affect one or more subunits of the GABA_(A) receptors such as α-subunits (GABRA1, GABRA2, GABRA3, GABRA4, GABRA5, GABRA6), β-subunits (GABRB1, GABRB2, GABRB3), γ-subunits (GABRG1, GABRG2, GABRG3), δ-subunits (GABRD), ε-subunits (GABRE), π-subunits (GABRP), θ-subunits (GABRQ), particularly GABARA5, GABRB3 and GABRG5. The T-type calcium channel antagonist can be selective for T-type calcium channel compared to inhibition of GABA receptors, e.g., having at least a 2-fold, at least a 5-fold, at least a 10-fold, at least a 20-fold, at least a 100-fold, at least a 500-fold or at least a 1000-fold selectivity (expressed, e.g., in terms of K_(i) or binding affinity).

In some embodiments, the T-type calcium channel antagonist can be one that does not cause one or more of the following side-effects or adverse events upon administration to animals, e.g., humans: liver damage, morphological changes in the animal liver, functional changes in the animal liver, kidney damage, morphological changes in the animal kidney, functional changes in the animal kidney, systemic lupus erythematosus, suicidal thoughts, suicidal behavior, suicidal ideation, increased risk of suicide, emergence or worsening of depression, unusual changes in mood or behavior, birth defects, allergic reaction.

In some embodiments, the T-type calcium channel antagonist can be one that does not cause one or more of the following side-effects or adverse events upon administration to animals: adverse events involving the gastrointestinal system such as anorexia, vague gastric upset, nausea and vomiting, cramps, epigastric and abdominal pain, weight loss, diarrhea, gum hypertrophy and swelling of the tongue; adverse events involving the hemopoietic system such as leukopenia, agranulocytosis, pancytopenia, with or without bone marrow suppression, and eosinophilia; adverse events involving the nervous system, including neurological reactions, sensory reactions, or psychiatric or psychological aberrations such as drowsiness, headache, dizziness, euphoria, hiccups, irritability, hyperactivity, lethargy, fatigue, ataxia, confusion, disturbances of sleep, night terrors, inability to concentrate, aggressiveness, paranoid psychosis, increased libido, or increased state of depression with overt suicidal intentions; adverse events involving the integumentary system including dermatologic manifestations such as urticaria, Stevens-Johnson syndrome, systemic lupus erythematosus, pruritic erythematous rashes, and hirsutism; adverse events involving the special senses such as myopia; and adverse events involving the genitourinary system, such as vaginal bleeding or microscopic hematuria.

In some embodiments, the T-type calcium channel antagonist is an antibody. Various methods for the preparation of antibodies are known in the art. See, Antibodies: A Laboratory Manual, CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989). For example, antibodies can be prepared by immunizing a suitable mammalian host with a sample of whole cells isolated from a patient. Antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies as described herein, or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies.

In some embodiments, the antibody is a monoclonal antibody. A “monoclonal antibody” is an antibody obtained from a population of substantially homogeneous antibodies, i.e., the antibodies comprising the population are identical except for possible naturally occurring mutations that are present in minor amounts.

In some embodiments, an antibody provided herein can be produced by recombinant means. In some embodiments, the antibody is a “humanized” or human antibody. “Humanized” or human antibodies can also be produced, and are preferred for use in therapeutic contexts. Methods for humanizing murine and other non-human antibodies, by substituting one or more of the non-human antibody sequences for corresponding human antibody sequences, are well known. See, e.g., Jones et al., Nature, 1986, 321, 522-25; Riechmann et al., Nature, 1988, 332, 323-27; Verhoeyen et al., Science, 1988, 239, 1534-36, Carter et al., Proc. Natl. Acad. Sci. USA, 1993, 89, 4285; and Sims et al., J. Immunol., 1993, 151, 2296. These humanized antibodies are designed to minimize unwanted immunological response toward rodent antihuman antibody molecules which limits the duration and effectiveness of therapeutic applications of those moieties in human recipients. Accordingly, preferred antibodies used in the therapeutic methods described herein are those that are either fully human or humanized with high affinity but exhibit low or no antigenicity in the subject.

In some embodiments, the T-type calcium channel antagonist is an oligonucleotide inhibitor. Example oligonucleotide inhibitors include, but are not limited to, antisense oligonucleotides, RNAi, dsRNA, siRNA and ribozymes. In some embodiments, the T-type calcium channel antagonist is a siRNA. As used in the specification, “antisense oligonucleotide” refers to a stretch of single-stranded DNA or RNA, usually chemically modified, whose sequence (3′-5′) is complementary to the sense sequence of a molecule of mRNA. Antisense molecules effectively inhibit gene expression by forming RNA/DNA duplexes. Antisense is understood to work by a variety of mechanisms, including physically blocking the ability of ribosomes to move along the messenger RNA, and hastening the rate at which the mRNA is degraded within the cytosol.

In order to avoid digestion by DNAse, antisense oligonucleotides can be chemically modified. For example, phosphorothioate oligodeoxynucleotides are stabilized to resist nuclease digestion by substituting one of the non-bridging phosphoryl oxygen of DNA with a sulfur moiety. Increased antisense oligonucleotide stability can also be achieved using molecules with 2-methoxyethyl (MOE) substituted backbones as described generally in U.S. Pat. No. 6,451,991, incorporated by reference, and U.S. Pat. Appl. Publ. No. 2003/0158143-A1. Thus, the antisense oligonucleotide can be modified to enhance in vivo stability relative to an unmodified oligonucleotide of the same sequence. The modification may be, e.g., a (2′-O-2-methoxyethyl) modification. The oligonucleotide may have a phosphorothioate backbone throughout, the sugar moieties of nucleotides 1-4 and 18-21 may bear 2′-O-methoxyethyl modifications and the remaining nucleotides may be 2′-deoxynucleotides.

It is understood in the art that an antisense oligonucleotide need not have 100% identity with the complement of its target sequence in order to be effective. The antisense oligonucleotides, therefore, can have a sequence that is at least about 70% identical to the complement of the target sequence. In one embodiment, the antisense oligonucleotides have can a sequence that is at least about 80% identical to the complement of the target sequence. In other embodiments, they have a sequence that is at least about 90% identical or at least about 95% identical to the complement of the target sequence, allowing for gaps or mismatches of several bases. Identity can be determined, for example, by using the BLASTN program of the University of Wisconsin Computer Group (GCG) software.

The antisense oligonucleotides according to the present invention are typically between 7 and 100 nucleotides in length. In one embodiment, the antisense oligonucleotides comprise from about 7 to about 50 nucleotides, or nucleotide analogues. In another embodiment, the antisense oligonucleotides comprise from about 7 to about 35 nucleotides, or nucleotide analogues. In other embodiments, the antisense oligonucleotides comprise from about 12 to about 35 nucleotides, or nucleotide analogues, and from about 15 to about 25 nucleotides, or nucleotide analogues.

The oligonucleotide inhibitors according to the present invention can be siRNA molecules that are targeted to a gene of interest such that the sequence of the siRNA corresponds to a portion of said gene. RNA molecules used in the present invention generally comprise an RNA portion and some additional portion, for example a deoxyribonucleotide portion.

The present disclosure further contemplates ribozyme oligonucleotide modulators that specifically target mRNA encoding a protein of interest, such as the proteins comprising the T-type calcium channel. Ribozymes are RNA molecules having an enzymatic activity that enables the ribozyme to repeatedly cleave other separate RNA molecules in a nucleotide-sequence specific manner. Such enzymatic RNA molecules can be targeted to virtually any mRNA transcript, and efficient cleavage can be achieved in vitro. Kim et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 8788; Haseloff et al., Nature, 1988, 334, 585; Cech, JAMA, 1988, 260, 3030; and Jefferies et al., Nucleic Acids Res., 1989, 17, 1371.

Typically, a ribozyme comprises two portions held in close proximity: an mRNA binding portion having a sequence complementary to the target mRNA sequence and a catalytic portion which acts to cleave the target mRNA. A ribozyme acts by first recognizing and binding a target mRNA by complementary base-pairing through the target mRNA binding portion of the ribozyme. Once it is specifically bound to its target, the ribozyme catalyzes cleavage of the target mRNA. Such strategic cleavage destroys the ability of a target mRNA to direct synthesis of an encoded protein. Having bound and cleaved its mRNA target, the ribozyme is released and can repeatedly bind and cleave new target mRNA molecules.

In some embodiments, the selective T-type calcium channel antagonist substantially crosses the blood brain barrier.

In some embodiments, the selective T-type calcium channel antagonist does not substantially cross the blood brain barrier.

In some embodiments, the T-type calcium channel antagonist is a calcium channel antagonist that selectively targets T-type calcium channels. In some embodiments, the T-type calcium channel antagonist is a small molecule as described herein.

In some embodiments, the T-type calcium channel antagonist selectively targets Cav3.1. In some embodiments, the T-type calcium channel antagonist selectively targets Cav3.2. In some embodiments, the T-type calcium channel antagonist selectively targets Cav3.3. “Selective” in this context means that the T-type calcium channel antagonist is more potent at antagonizing one type of T-type calcium channel over another type of calcium channel, e.g., more potent at antagonizing Cav3.1 than Cav3.2 or Cav3.3 or both; more potent at antagonizing Cav3.2 than Cav3.1 or Cav3.3 or both; more potent at antagonizing Cav3.3 than Cav3.1 or Cav3.2 or both. Selectivity can be determined, e.g., by comparing the IC₅₀ of a compound in inhibiting one type of T-type calcium channel with its IC₅₀ in inhibiting the other types of T-type calcium channel: if the IC₅₀ for inhibiting one type of T-type channels is lower than the IC₅₀ for inhibiting the other type of T-type calcium channel, the compound is considered selective. An IC₅₀ ratio of 0.1 (or lower) denotes 10-fold (or greater) selectivity. An IC₅₀ ratio of 0.01 (or lower) denotes 100-fold (or greater) selectivity. An IC₅₀ ratio of 0.001 (or lower) denotes 1000-fold (or greater) selectivity. In some embodiments, the selectivity for Cav3.1, Cav3.2 or Cav3.3 is 10-fold or greater, 100-fold or greater, or 1000-fold or greater.

In some embodiments, the T-type calcium channel antagonist selectively targets T-type calcium channels (e.g., Cav3.1, Cav3.2, and/or Cav3.3) over sodium channels such as sodium channels having Nav 1.1, Nav 1.2, Nav 1.3, Nav 1.4, Nav 1.5, Nav 1.6, Nav 1.7, Nav 1.8, or Nav 1.9 alpha subunits, and/or Nav β1, Nav β2, Nav β3, Nav β4 subunits. The T-type calcium channel antagonist can be selective for T-type calcium channel compared to inhibition of sodium channels. Selectivity can be determined, e.g., by comparing the IC₅₀ of a compound in inhibiting one or more of the types of T-type calcium channel with its IC₅₀ in inhibiting the one or more types of sodium channel: if the IC₅₀ for inhibiting the T-type calcium channels is lower than the IC₅₀ for inhibiting the sodium channel, the compound is considered selective. An IC₅₀ ratio of 0.1 (or lower) denotes 10-fold (or greater) selectivity. An IC₅₀ ratio of 0.01 (or lower) denotes 100-fold (or greater) selectivity. An IC₅₀ ratio of 0.001 (or lower) denotes 1000-fold (or greater) selectivity. In some embodiments, the selectivity for T-type calcium channels is 10-fold or greater, 100-fold or greater, or 1000-fold or greater.

The effectiveness of a compound in inhibiting T-type calcium channels may vary depending on the state of the T-type calcium channel that the T-type calcium channel antagonist inhibits. T-type calcium channels can occur in different states depending on the cell membrane potential. T-type calcium channel antagonists that are effective in the methods described herein may include T-type calcium channel antagonists that block T-type calcium channels when the membrane potential is in the range from about −60 mV to about −30 mV, e.g., preferably about −40 mV. A membrane potential “in the range from about −60 to about −30 mV” can include membrane potentials within a range of −70 mV to −20 mV, or within a range of −65 mV to −25 mV, and can also encompass membrane potential ranges such as about −40 mV to about −30 mV, about −50 mV to about −30 mV, about −70 mV to about −30 mV, about −50 mV to about −40 mV, about −60 mV to about −40 mV, about −70 mV to about −40 mV, about −60 mV to about −50 mV, and about −70 to about −50 mV, as well as about −30 mV, about −40 mV, about −50 mV, and about −60 mV. In some embodiments, the T-type calcium channel antagonists that are effective in the methods described herein may include T-type calcium channel antagonists that block T-type calcium channels when the membrane potential is in the range from about −100 mV to about −80 mV, e.g., preferably about −90 mV. A membrane potential “in the range from about −100 to about −80 mV” can include membrane potentials within a range of −110 mV to −70 mV, or within a range of −105 mV to −75 mV, and can also encompass membrane potential ranges such as about −100 mV to about −80 mV, about −90 mV to about −80 mV, and about −100 mV to about −90 mV, as well as about −100 mV, about −90 mV, and about −80 mV.

While not being limited by any theory, it is believed that T-type calcium channel antagonists that are effective in the methods described herein may include T-type calcium channel antagonists that block T-type calcium channels when the membrane potential is in the range from about −60 mV to about −30 mV, e.g., about −40 mV selectively when compared to blockade of the T-type calcium channels when the membrane potential is in the range from about −100 mV to about −80 mV, e.g., about −90 mV.

A T-type channel inhibitor that is effective may inhibit T-type calcium channels with an IC₅₀ for inhibiting T-type calcium channels when the membrane potential is about −40 mV that is about 10 μM or lower, e.g., about 1 μM or lower, about 500 nM or lower, about 100 nM or lower, about 50 nM or lower, about 10 nM or lower, about 5 nM or lower, or about 1 nM or lower. A T-type calcium channel antagonist that is effective may inhibit T-type calcium channels at a membrane potential of about −40 mV selectively compared to inhibition of T-type calcium channels at a membrane potential of about −90 mV. For example, the ratio of the IC₅₀ of the T-type calcium channel antagonist in inhibiting T-type calcium channels at a membrane potential of about −40 mV selectively compared to inhibition of T-type calcium channels at a membrane potential of about −90 mV may be about 1:2 or lower, e.g., about 1:5 or lower, about 1:10 or lower, about 1:20 or lower, about 1:50 or lower, about 1:100 or lower, about 1:500 or lower, about 1:1000 or lower. In some embodiments, the selectivity for inhibiting T-type calcium channels at about −40 mV compared to inhibiting T-type calcium channels at about −90 mV is 2-fold or greater, 5-fold or greater, 10-fold or greater, 100-fold or greater, or 1000-fold or greater.

A T-type channel inhibitor that is effective may inhibit T-type calcium channels with an IC₅₀ for inhibiting T-type calcium channels when the membrane potential is about −90 mV that is about 10 μM or lower, e.g., about 1 μM or lower, about 500 nM or lower, about 100 nM or lower, about 50 nM or lower, about 10 nM or lower, about 5 nM or lower, or about 1 nM or lower. A T-type calcium channel antagonist that is effective may inhibit T-type calcium channels at a membrane potential of about −90 mV selectively compared to inhibition of T-type calcium channels at a membrane potential of about −40 mV. For example, the ratio of the IC₅₀ of the T-type calcium channel antagonist in inhibiting T-type calcium channels at a membrane potential of about −90 mV selectively compared to inhibition of T-type calcium channels at a membrane potential of about −40 mV may be about 1:2 or lower, e.g., about 1:5 or lower, about 1:10 or lower, about 1:20 or lower, about 1:50 or lower, about 1:100 or lower, about 1:500 or lower, about 1:1000 or lower. In some embodiments, the selectivity for inhibiting T-type calcium channels at about −90 mV compared to inhibiting T-type calcium channels at about −40 mV is 2-fold or greater, 5-fold or greater, 10-fold or greater, 100-fold or greater, or 1000-fold or greater.

All compounds, and pharmaceutically acceptable salts thereof, can be found together with other substances such as water and solvents (e.g. hydrates and solvates) or can be isolated. In some embodiments, the compounds provided herein, or pharmaceutically acceptable salts thereof, are substantially isolated. By “substantially isolated” is meant that the compound is at least partially or substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the compounds provided herein. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the compounds provided herein, or salt thereof. Methods for isolating compounds and their salts are routine in the art.

As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the present application include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present application can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethyl acetate, alcohols (e.g., methanol, ethanol, iso-propanol, or butanol) or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977). Methods for preparing salt forms are described, for example, in Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH, 2002.

IV. Combination Therapies

One or more additional therapeutic agents can be used in combination with the compounds provided herein for the treatment of Angelman Syndrome or Prader-Willi syndrome. Example additional therapeutic agents include, but are not limited to calcium channel antagonists (including L-type and T-type antagonists), anticonvulsant agents, GABA(A) receptor agonists and positive allosteric modulators or gene therapy or gene reactivation therapy (Bailus et al., The prospect of molecular therapy for Angelman syndrome and other monogenic neurologic disorders, BMC Neuroscience, 2014, 15, 76).

In some embodiments, the treatment with the T-type calcium channel antagonist can be provided in the absence of additional pharmacological agents for treating Angelman Syndrome or Prader-Willi syndrome. In some embodiments, the treatment can be performed with a single T-type calcium channel antagonist. In some embodiments, the treatment with the T-type calcium channel antagonist can be provided in the absence of an anticonvulsant agent.

The one or more additional pharmaceutical agents can be administered to a patient simultaneously or sequentially, using the same schedule or a different schedule of administration, which will be determined by the particular combination used and the judgment of the prescribing physician.

Example calcium channel antagonists include, but are not limited to, the T-type calcium channel antagonists described herein, and L-type calcium channel antagonists. In some embodiments, the additional calcium channel antagonist is selected from a T-type calcium channel antagonist provided herein. In some embodiments, the additional calcium channel antagonist is an L-type calcium channel antagonist. In some embodiments, the additional calcium channel antagonist is a T-type calcium channel antagonist. In some embodiments, the additional calcium channel antagonist is a T-type calcium channel antagonist selected from the group consisting of mibefradil, MK-5395, MK-6526, MK-8998, and Z944. In some embodiments, the additional calcium channel antagonist is a T-type calcium channel antagonist and an L-type calcium channel antagonist. In some embodiments, the additional calcium channel antagonist is a T-type calcium channel antagonist or an L-type calcium channel antagonist selected from the group consisting of ACT-28077, mibefradil, and TTL-1177. In some embodiments, the additional calcium channel antagonist is mibefradil.

Example anticonvulsant agents include, but are not limited to, acetazolamide, carbamazepine, clobazam, clonazepam, eslicarbazepine acetate, ethosuximide, gabapentin, lacosamide, lamotrigine, levetiracetam, nitrazepam, oxcarbazepine, perampanel, piracetam, phenobarbital, phenytoin, pregabalin, primidone, retigabine, rufinamide, valproate, e.g., sodium valproate, stiripentol, tiagabine, topiramate, vigabatrin, and zonisamide.

Example GABA(A) receptor agonists include gaboxadol, bamaluzole, gamma-aminobutyric acid, gabamide, gamma-amino-beta-hydroxybutyric acid, gaboxadol, ibotenic acid, isoguvacine, isonipecotic acid, muscimol, phenibut, picamilon, progabide, quisqualamine, SL 75102, and thiomuscimol.

Example GABA(A) receptor positive allosteric modulators include avermectins (e.g., ivermectin), barbiturates (e.g., phenobarbital), benzodiazepines (e.g., adinazolam, alprazolam, bentazepam, bretazenil, bromazepam, brotizolam, camazepam, chlordiazepoxide, cinazepam, cinolazepam, clobazam, clonazepam, clonazolam, clorazepate, clotiazepam, cloxazolam, delorazepam, diazepam, diclazepam, estazolam, ethyl carfluzepate, etizolam, ethyl loflazepate, flubromazepam, flubromazolam, flunitrazepam, flurazepam, flutazolam, flutoprazepam, halazepam, ketazolam, loprazolam, lorazepam, lormetazepam, medazepam, mexazolam, midazolam, nifoxipam, nimetazepam, nitrazepam, nordiazepam, oxazepam, phenazepam, pinazepam, prazepam, premazepam, pyrazolam, quazepam, rilmazafone, temazepam, thienalprazolam, tetrazepam, and triazolam), bromides (e.g., potassium bromide, carbamates (e.g., meprobamate, carisoprodol), chloralose, chlormezanone, clomethiazole, dihydroergolines (e.g., ergoloid (dihydroergotoxine)), etazepine, etifoxine, Imidazoles (e.g., etomidate), kavalactones (found in kava), loreclezole, neuroactive steroids (e.g., allopregnanolone, ganaxolone), nonbenzodiazepines (e.g., zaleplon, zolpidem, zopiclone, eszopiclone), petrichloral, phenols (e.g., propofol), piperidinediones (e.g., glutethimide, methyprylon), propanidid, pyrazolopyridines (e.g., etazolate), quinazolinones (e.g., methaqualone), skullcap constituents, stiripentol, sulfonylalkanes (e.g., sulfonmethane, tetronal, trional), and valerian constituents (e.g., valeric acid, valerenic acid).

In some embodiments, the therapy can be administered as a monotherapy. In some embodiments, the therapy can be administered in the absence of additional antiepileptic therapy. The therapy can be administered in the absence of any of the additional agents described in this section. For example, the therapy can be administered in the absence of additional anticonvulsant such as, acetazolamide, carbamazepine, clobazam, clonazepam, eslicarbazepine acetate, ethosuximide, gabapentin, lacosamide, lamotrigine, levetiracetam, nitrazepam, oxcarbazepine, perampanel, piracetam, phenobarbital, phenytoin, pregabalin, primidone, retigabine, rufinamide, valproate, e.g., sodium valproate, stiripentol, tiagabine, topiramate, vigabatrin, or zonisamide.

Example gene reactivation therapies include administration of compounds that activate the paternal (or maternal) copy of UBE3A. Examples include topoisomerase inhibitors (including topoisomerase I and II inhibitors) such as topotecan, irinotecan, etoposide and dexrazoxane, and other compounds identified by Huang et al., “Topoisomerase inhibitors unsilence the dormant allele of Ube3a in neurons”, Nature, 2011, 481, 185-89.

In some embodiments, the T-type calcium channel antagonists provided herein can be used in combination with one or more additional therapies including, but not limited to, physical therapy, occupational therapy, communication therapy, and behavioral therapy. In some embodiments, the T-type calcium channel antagonists provided herein can be used in combination with one or more additional therapeutic agents and one or more additional therapies selected from the group consisting of physical therapy, occupational therapy, communication therapy, and behavioral therapy.

V. Pharmaceutical Compositions

The T-type calcium channel inhibitors used in the methods described herein can be administered in the form of pharmaceutical compositions. Thus the present disclosure provides T-type calcium channel inhibitor, and at least one pharmaceutically acceptable carrier for use in the claimed methods of treatment, or the manufacture of a medicament for treating conditions as described herein. These compositions can be prepared in a manner known in the pharmaceutical art, and can be administered by a variety of routes. Administration may be topical (including transdermal, epidermal, ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal or intranasal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal intramuscular or injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Parenteral administration can be in the form of a single bolus dose, or may be, e.g., by a continuous perfusion pump. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

This pharmaceutical compositions which contain, as the active ingredient, a T-type calcium channel inhibitor (which can be in the form of a pharmaceutically acceptable salt), in combination with one or more pharmaceutically acceptable carriers (excipients). In some embodiments, the composition is suitable for topical administration. In making the compositions of the invention, the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, e.g., a capsule, sachet, paper, or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, e.g., up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders.

In preparing a formulation, the T-type calcium channel inhibitor can be milled to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it can be milled to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size can be adjusted by milling to provide a substantially uniform distribution in the formulation, e.g., about 40 mesh.

The compounds of the invention may be milled using known milling procedures such as wet milling to obtain a particle size appropriate for tablet formation and for other Formulation types. Finely divided (nanoparticulate) preparations of the compounds of the invention can be prepared by processes known in the art.

Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup and methyl cellulose. The Formulations can additionally include: lubricating agents such as talc, magnesium stearate and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents.

In some embodiments, the pharmaceutical composition comprises silicified microcrystalline cellulose (SMCC) and at least one compound described herein, or a pharmaceutically acceptable salt thereof. In some embodiments, the silicified microcrystalline cellulose comprises about 98% microcrystalline cellulose and about 2% silicon dioxide wt/wt.

In some embodiments, a wet granulation process is used to produce the composition. In some embodiments, a dry granulation process is used to produce the composition.

The compositions can be formulated in a unit dosage form, each dosage containing from about 5 to about 1,000 mg (1 g), more usually about 100 mg to about 500 mg, of the active ingredient. In some embodiments, each dosage contains about 10 mg of the active ingredient. In some embodiments, each dosage contains about 50 mg of the active ingredient. In some embodiments, each dosage contains about 25 mg of the active ingredient. The term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.

The components used to formulate the pharmaceutical compositions are of high purity and are substantially free of potentially harmful contaminants (e.g., at least National Food grade, generally at least analytical grade, and more typically at least pharmaceutical grade). Particularly for human consumption, the composition is preferably manufactured or Formulated under Good Manufacturing Practice standards as defined in the applicable regulations of the U.S. Food and Drug Administration. For example, suitable Formulations may be sterile and/or substantially isotonic and/or in full compliance with all Good Manufacturing Practice regulations of the U.S. Food and Drug Administration.

The active compound may be effective over a wide dosage range and is generally administered in a therapeutically effective amount. It will be understood, however, that the amount of the compound actually administered will usually be determined by a physician, according to the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms and the like.

The therapeutic dosage of a compound of the present invention can vary according to, e.g., the particular use for which the treatment is made, the manner of administration of the compound, the health and condition of the patient, and the judgment of the prescribing physician. The proportion or concentration of a compound of the invention in a pharmaceutical composition can vary depending upon a number of factors including dosage, chemical characteristics (e.g., hydrophobicity), and the route of administration. For example, the compounds of the invention can be provided in an aqueous physiological buffer solution containing about 0.1 to about 10% w/v of the compound for parenteral administration. Some typical dose ranges are from about 1 μg/kg to about 1 g/kg of body weight per day. In some embodiments, the dose range is from about 0.01 mg/kg to about 100 mg/kg of body weight per day. The dosage is likely to depend on such variables severity of the disease, the overall health status of the particular patient, the relative biological efficacy of the compound selected, Formulation of the excipient, and its route of administration. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems. Effective doses for a human can be, e.g., about 1 mg, 2 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 110 mg, 120 mg, 125 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg or 1000 mg. The doses can be administered, e.g., once a day, twice a day, three times a day, or four times a day.

In some embodiments, when the T-type calcium channel antagonist is mibefradil, and the mibefradil can be administered at a dose of, e.g., about 0.1 mg, 0.3 mg, 1 mg, 3 mg, 5 mg, 10 mg. 15 mg. or 30 mg. The doses can be administered, e.g., once a day, twice a day, three times a day, or four times a day.

In some embodiments, when the T-type calcium channel antagonist is MK-5395, and the MK-5395 can be administered at a dose of, e.g., about 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg. 30 mg/kg, or 100 mg/kg. The doses can be administered, e.g., once a day, twice a day, three times a day, or four times a day.

In some embodiments, the T-type calcium channel antagonist is MK-6526, and the MK-6526 can be administered at a dose of, e.g., about 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg. 30 mg/kg, or 100 mg/kg. The doses can be administered, e.g., once a day, twice a day, three times a day, or four times a day.

In some embodiments, the T-type calcium channel antagonist is MK-8998, and the MK-8998 can be administered at a dose of, e.g., about 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg. 30 mg/kg, or 100 mg/kg. The doses can be administered, e.g., once a day, twice a day, three times a day, or four times a day.

In some embodiments, the T-type calcium channel antagonist is Z944, and the Z944 can be administered at a dose of, e.g., about 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg. 30 mg/kg, or 100 mg/kg. The doses can be administered, e.g., once a day, twice a day, three times a day, or four times a day.

For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention. When referring to these preformulation compositions as homogeneous, the active ingredient is typically dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation is then subdivided into unit dosage forms of the type described above containing from, e.g., about 0.1 to about 1000 mg of the active ingredient of the present invention.

The liquid forms in which the compounds and compositions of the present invention can be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described supra. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions can be nebulized by use of inert gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device can be attached to a face mask, tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions can be administered orally or nasally from devices which deliver the Formulation in an appropriate manner.

Topical formulations can contain one or more carriers. In some embodiments, ointments can contain water and one or more hydrophobic carriers selected from, e.g., liquid paraffin, polyoxyethylene alkyl ether, propylene glycol, white petroleum jelly, and the like. Carrier compositions of creams can be based on water in combination with glycerol and one or more other components, e.g., glycerinemonostearate, PEG-glycerinemonostearate and cetylstearyl alcohol. Gels can be formulated using isopropyl alcohol and water, suitably in combination with other components such as, e.g., glycerol, hydroxyethyl cellulose, and the like. In some embodiments, topical formulations contain at least about 0.1, at least about 0.25, at least about 0.5, at least about 1, at least about 2 or at least about 5 wt % of the compound of the invention.

The amount of compound or composition administered to a patient will vary depending upon what is being administered, the purpose of the administration, such as prophylaxis or therapy, the state of the patient, the manner of administration and the like. In therapeutic applications, compositions can be administered to a patient already suffering from a disease in an amount sufficient to eliminate or at least partially alleviate the symptoms of the disease and its complications. Effective doses will depend on the disease condition being treated as well as by the judgment of the attending clinician depending upon factors such as the severity of the disease, the age, weight and general condition of the patient and the like.

The compositions administered to a patient can be in the form of pharmaceutical compositions described above. These compositions can be sterilized by conventional sterilization techniques, or may be sterile filtered. Aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the compound preparations typically will be between 3 and 11, more preferably from 5 to 9 and most preferably from 7 to 8. It will be understood that use of certain of the foregoing excipients, carriers or stabilizers will result in the formation of pharmaceutical salts.

The therapeutic dosage of a T-type calcium channel antagonist used in the methods described herein can vary according to, e.g., the particular use for which the treatment is made, the manner of administration of the compound, the health and condition of the patient, and the judgment of the prescribing physician. The proportion or concentration of a compound of the invention in a pharmaceutical composition can vary depending upon a number of factors including dosage, chemical characteristics (e.g., hydrophobicity), and the route of administration. For example, the T-type calcium channel antagonists can be provided in an aqueous physiological buffer solution containing about 0.1 to about 10% w/v of the compound for parenteral administration. Some typical dose ranges are from about 1 μg/kg to about 1 g/kg of body weight per day. In some embodiments, the dose range is from about 0.01 mg/kg to about 100 mg/kg of body weight per day. The dosage is likely to depend on such variables as the type and extent of progression of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the compound selected, formulation of the excipient, and its route of administration. Effective doses can be extrapolated from dose-response curves derived from in vitro or in vivo model test systems.

EXAMPLES

The invention is further described in the following example, which does not limit the scope of the invention defined in the claims.

Example 1. Treatment of a Patient Having Angelman Syndrome

A patient has Angelman Syndrome, which is characterized by mental disability and frequent erratic movement (e.g., seizures, hand-flapping). The patient is administered, alone or in combination with other therapies, a therapeutically effective amount of a T-type calcium channel antagonist provided herein (e.g., mibefradil, efonidipine, TTL-1177, nickel, and the like). After a sufficient dosage of the T-type calcium channel antagonist has been administered (e.g., after one dose or after a series of doses), the patient's brain activity is monitored (e.g., using an electroencephalogram (EEG)). The patient's brain activity demonstrates measurable improvement in Angelman Syndrome-characteristic brain activity compared to the brain activity of the patient prior to treatment with the T-type calcium channel antagonist. The patient may also exhibit improvements in other symptoms, including intellectual and developmental disability, sleep disturbance, seizures, and jerky movements.

Example 2. In Vivo Study in Murine Angelman Model

Three separate tests were performed to determine effects of two compounds, mibefradil and MK-8998 on behavioral and physiological deficits and epilepsy in a mouse model for Angelman syndrome. The tests included: audiogenic-induced epilepsy test (Test 1, T1), a battery of motor/behavioral tests (Test 2, T2) and long-term potentiation measurements (LTP, Test 3, T3). Completion of each test provided a quantitative result of mibefradil or MK-8998 effect on this test. For all three tests, 2 separate groups of mice were generated: group 1 for test 1 and group 2 for tests 2 and 3, as shown below in see Table 2 and each group consists of 6 subgroups, a-f.

For all behavioral and electrophysiology experiments female Ube3am⁻/p⁺KO mice (i.e., AS mice) were crossed with wild-type males, to generate heterozygous AS mice and littermate controls in the F1 hybrid 12952-C57BL/6 background. For epilepsy tests, AS mice are crossed with controls in the 12952/SvPasCrl background. In total, 2 groups of mice are required: group 1 for the epilepsy test (Table 2, group 1a-f) and group 2 for the behavioral test battery and electrophysiology (Table 2, group 2a-f). Each group consists of 6 subgroups, including 2 control subgroups: wild-type (a) and AS mutants (b) mice on standard diet, and 4 subgroups on drug-supplemented diets: Mibefradil (c-d) and MK-8998 (e-f) groups each at two different concentrations. The number of mice used per test/subgroup is indicated in Table 2 and ranges from 5-7 mice/subgroup for electrophysiology, 10 mice/subgroup for epilepsy test, and 12 to 15 mice/subgroup for behavioral test battery. Adult male and female mice (age <8 weeks) for were used for all experiments.

TABLE 2 Group/ N/ Model/ Test subgroup group subgroup Treatment T1 1a 10 Wildtype Standard chow T1 1b 10 Ube3a^(stop/p+) Standard chow T1 1c 10 Ube3a^(stop/p+) Mibefradil T1 1d 10 Ube3a^(stop/p+) Mibefradil T1 1e 10 Ube3a^(stop/p+) CX-8998 T1 1f 10 Ube3a^(stop/p+) CX-8998 T2-3* 2a  12-15** Wildtype Standard chow T2-3 2b 12-15 Ube3a^(stop/p+) Standard chow T2-3 2c 12-15 Ube3a^(stop/p+) Mibefradil T2-3 2d 12-15 Ube3a^(stop/p+) Mibefradil T2-3 2e 12-15 Ube3a^(stop/p+) CX-8998 T2-3 2f 12-15 Ube3a^(stop/p+) CX-8998 *Test 2 performed in two batches of 7-8 mice/subgroup/batch. **for behavioral test battery: at minimum 12 mice/subgroup tested and 15 mice/subgroup for statistical power.

Mibefradil and MK-8998 were tested at various different doses (e.g., 10 mg/kg, 30 mg/kg, 40 mg/kg, 60 mg/kg) based on the assumption that mice eat 5 g chow/day. Both drugs were administered in chow starting after the weaning of the pups (P21; subgroups c-f). Control mice (subgroup a and b) received standard chow.

Effect of Mibefradil and MK-8998 on Epilepsy in Angelman Syndrome (AS) Mouse Model (Test 1)

AS-mutant mice, wild-type, and AS mutant mice were maintained on either standard chow (group 1a and Ib), or on drug-supplemented chow (group Ic-f) from postnatal day 21 (P21) until adulthood. At age of 8 weeks (P56) all mice were tested for occurrence of audiogenic-induced seizures. The outcome of this test provided a number (or %) of mice/subgroup which experienced epilepsy. Most (>90%) AS-mutant mice on standard diet (group Ib) were expected to experience epilepsy. In contrast, wild-type mice on standard diet (group 1a) were not expected to show seizures (<15%) (see e.g., Silva-santos et al., J. Clin. Invest., 2015, 125(5), 2069-2076). A brief summary of the Test 1 protocol is shown below:

1. Epilepsy Assessment: (12952/SvPasCrl×AS Mice)

a. 9 mice/group

b. 6 groups as follows:

-   -   i. Vehicle: Wild type     -   ii. Mibefradil (Oral gavage)         -   1. 10 mg/kg         -   2. 40 mg/kg     -   iii. MK-8998 (Oral gavage)         -   1. 10 mg/kg         -   2. 30 mg/kg         -   3. 60 mg/kg

c. Treatment schedule: Baseline epilepsy test→Begin 7 days of treatment, P56→Epilepsy test

d. Results: % of mice to experience audiogenic-induced epilepsy

First, the mice underwent the epilepsy test prior to treatment with the mibefradil or MK-8998. The mice were then treated with either mibefradil or MK-8998 via oral gavage for 7 days and subsequently underwent the epilepsy test. The mice were dosed as follows: Group 1: mibefradil 10 mg/kg and 40 mg/kg; MK-8998: 10 mg/kg and 30 mg/kg. Group 2: mibefradil 10 mg/kg and 40 mg/kg; MK-8998: 30 mg/kg and 60 mg/kg. Results of the audiogenic-induced epilepsy tests are shown in FIG. 1.

The results show that, remarkably, MK-8998 eliminated seizures in a dose-dependent manner, with seizures being eliminated completely in about 80% of treated animals at 60 mg/kg. It is important to note, however, that the absence of an apparent response in the data shown in FIG. 1 does not show that treatment was ineffective since FIG. 1 only records the number of seizure free animals recorded. An animal could have a reduced number of seizures or reduced severity of seizures, yet still be recorded as having seizures in the data shown in FIG. 1.

It is expected that the seizure-prone animals treated with mibefradil or MK-8998 or other T-type calcium channel inhibitor exhibit a reduced number of seizures, or a reduced severity of seizures, or both.

Test 2. Effect of Mibefradil and MK-8998 on Behavioral Deficits in AS-Mice

A battery of well-established motor coordination and behavioral tests in which AS-mutant mice show a robust phenotype (Silva-santos et al., J. Clin. Invest., 2015, 125(5), 2069-2076) and which includes rotarod, open field, marble burying, nest building and forced swim test will be carried out. For this tests battery a group consisting of 12-15 mice/subgroup (6 subgroups, see Table 2) will be tested resulting in a group of 72-90 mice. Due to limitations in breeding and testing capacity, this group will be split into two batches consisting of 7-8 mice/subgroup (42-48 mice/batch). Mice from both batches will undergo the same battery of motor/behavioral tests and results from both groups will be combined to reach statistical power. After each test (e.g. rotarod) a quantifiable result (outcome measure) will be obtain for each subgroup. A brief summary of the Test 2 protocol is shown below:

2. Behavioral Assessment: Heterozygous AS Mice (Male Wild Type×Female Ube3am−/p+KO Mice)

a. 12-15 mice/group

b. 6 groups as follows:

-   -   i. Vehicle: Wild type     -   ii. Vehicle: Heterozygous AS mouse     -   iii. Mibefradil         -   1. 10 mg/kg         -   2. 40 mg/kg     -   iv. MK-8998         -   1. 30 mg/kg         -   2. 60 mg/kg

c. Treatment schedule: Testing completed in 2 batches of 6 weeks of testing

d. Assessments and Results:

-   -   i. Accelerating Rotarod: Latency (s)     -   ii. Marble Burying: Number of Marbles Unburied     -   iii. Open Field Test: Path length (m)     -   iv. Nest Building: material used (%)     -   v. Forced Swim Test: Floating time (%).         Test 3. Effect of Mibefradil and MK-8998 on Synoptic Plasticity         Deficits in AS Mice.

Subsequently, the effect of mibefradil and MK-8998 on long term potentiation (LTP) deficit in AS-mutant mice will be tested. For this experiments, mice which underwent Test 2 can be used. LTP will be measured in 5-7 mice/subgroup. If required, a higher number of mice can be tested (e.g., from the second batch of mice) A brief summary of the Test 3 protocol is shown below:

3. Long Term Potentiation Assessment: Heterozygous AS Mice (Male Wild Type×Female Ube3am−/p+KO Mice)

a. 6-7 mice/group

b. 6 groups as follows:

-   -   i. Vehicle: Wild type     -   ii. Vehicle: Heterozygous AS mouse     -   iii. Mibefradil         -   1. 10 mg/kg         -   2. 40 mg/kg     -   iv. MK-8998         -   1. 30 mg/kg         -   2. 60 mg/kg

c. Treatment schedule: 2 mice per day. 5 weeks total

d. Results:

-   -   i. LTP induction and expression (% of baseline) in wild-type         mice     -   ii. Potential of a drug to rescue LTP-deficit in AS mice (% of         baseline)

Other Embodiments

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other aspects, advantages, embodiments and modifications are within the scope of the following claims. 

What is claimed is:
 1. A method of reducing or ameliorating seizure associated with Angelman Syndrome, said method comprising administering to a subject in need thereof a therapeutically effective amount of a T-type calcium channel antagonist, wherein the T-type calcium channel antagonist is MK-8998:

or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1, wherein said T-type calcium channel antagonist is MK-8998:


3. The method of claim 1, further comprising administering to the subject an additional therapeutic agent.
 4. The method of claim 3, wherein the additional therapeutic agent is an additional T-type calcium channel inhibitor.
 5. The method of claim 3, wherein the additional therapeutic agent is an anticonvulsive agent.
 6. The method of claim 1, further comprising administering an additional therapy selected from the group consisting of physical therapy, occupational therapy, communication therapy, and behavioral therapy.
 7. The method of claim 1, wherein the reducing or ameliorating seizure associated with Angelman Syndrome comprises reducing the frequency of seizure in the subject in need thereof.
 8. The method of claim 1, wherein the reducing or ameliorating seizure associated with Angelman Syndrome comprises reducing the severity of seizure in the subject in need thereof.
 9. The method of claim 1, wherein the reducing or ameliorating seizure associated with Angelman Syndrome comprises reducing the frequency and severity of seizure in the subject in need thereof. 